Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/61393
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Type: Journal article
Title: Probing the catalytic roles of Arg548 and Gln552 in the carboxyl transferase domain of the rhizobium etli pyruvate carboxylase by site-directed mutagenesis
Author: Duangpan, S.
Jitrapakdee, S.
Adina-Zada, A.
Byrne, L.
Zeczycki, T.
Maurice, M.
Cleland, W.
Wallace, J.
Attwood, P.
Citation: Biochemistry, 2010; 49(15):3296-3304
Publisher: Amer Chemical Soc
Issue Date: 2010
ISSN: 0006-2960
1520-4995
Statement of
Responsibility: 
Saowapa Duangpan, Sarawut Jitrapakdee, Abdussalam Adina-Zada, Lindsay Byrne, Tonya N. Zeczycki, Martin St. Maurice, W. Wallace Cleland, John C. Wallace and Paul V. Attwood
Abstract: The roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase were investigated using site-directed mutagenesis. Mutation of Arg548 to alanine or glutamine resulted in the destabilization of the quaternary structure of the enzyme, suggesting that this residue has a structural role. Mutations R548K, Q552N, and Q552A resulted in a loss of the ability to catalyze pyruvate carboxylation, biotin-dependent decarboxylation of oxaloacetate, and the exchange of protons between pyruvate and water. These mutants retained the ability to catalyze reactions that occur at the active site of the biotin carboxylase domain, i.e., bicarbonate-dependent ATP cleavage and ADP phosphorylation by carbamoyl phosphate. The effects of oxamate on the catalysis in the biotin carboxylase domain by the R548K and Q552N mutants were similar to those on the catalysis of reactions by the wild-type enzyme. However, the presence of oxamate had no effect on the reactions catalyzed by the Q552A mutant. We propose that Arg548 and Gln552 facilitate the binding of pyruvate and the subsequent transfer of protons between pyruvate and biotin in the partial reaction catalyzed in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase.
Keywords: Rhizobium etli
Biotin
Multiprotein Complexes
Pyruvate Carboxylase
Arginine
Glutamine
Bacterial Proteins
Magnetic Resonance Spectroscopy
Mutagenesis, Site-Directed
Binding Sites
Catalytic Domain
Protein Conformation
Protein Structure, Quaternary
Kinetics
Catalysis
Models, Molecular
Rights: Copyright © 2010 American Chemical Society
DOI: 10.1021/bi901894t
Grant ID: http://purl.org/au-research/grants/arc/DP0988153
Appears in Collections:Aurora harvest 5
Molecular and Biomedical Science publications

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